Heparin is a glycosaminoglycan, an acidic mucopolysaccharide composed of D-glucuronic acid and D-glucosamine with a high degree of N-sulphation. It is present in the form of proteoglycan in many mammalian tissues, such as the intestine, liver, lung, being localized in the connective tissue-type mast cells, which line for example the vascular and serosal system of mammals. The main pharmaceutical characteristic of heparin is its ability to enhance the activity of the natural anticoagulant, antithrombin III.
Heparins exist naturally bound to proteins, forming so called heparin proteoglycans. Usually, the endogenous or native, naturally existing heparin proteoglycans contain 10-15 heparin glycosaminoglycan chains, each chain having a molecular weight in the range of 75±25 kDa, and being bound to one core protein or polypeptide. Each native heparin glycosaminoglycan chain contains several separate heparin units consecutively placed end-to-end, which are cleaved by endoglycosidases in their natural environment. The natural or native conjugates are difficult to prepare in pure form. Thus, they have not been suggested for therapeutical or corresponding use. Heparin glycosaminoglycans belong to a larger group of negatively charged heteropolysaccharides, which generally are associated with proteins forming so called proteoglycans. Examples of other naturally existing glycosaminoglycans are for example chondroitin-4- and 6-sulphates, keratan sulphates, dermatan sulphates, hyaluronic acid, heparan sulphates and heparins. Of said heparin-like compounds existing in nature, only hyaluronic acid is generally not associated with a proteinaceous core molecule.
During the past decades the trend in heparin research has been to develop and use heparin chain units, which have been fractionated for systemic clinical preparations of shorter chains to increase specificity. The generally used two types of standard clinical heparins are the so called unfractionated or high-molecular weight heparins and fractionated or low-molecular-weight heparins. Said two types of heparins have an average molecular weight of 15 and 5 kDa, respectively. In the present invention these two types of heparins are both considered to be lower-molecular-weight heparins. Most commercial preparations have a molecular weight between 4 to 20 kDa depending on their origin, the method of preparation and/or determination. Thus, the commercial heparins belong to the lower-molecular-weight heparins as defined in the present invention.
In the U.S. Pat. No. 5,529,986 a synthetic macromolecular heparin conjugate is described. It consists of at least 20 heparin moieties, but can contain more than 100 heparin moieties, combined with natural or synthetic substantially straight-chained polymer backbones such as polylysine, polyornithine, chitosan, polyamine or polyallyl.
However, in said patent each heparin moiety is also characterized by a low molecular weight of approximately 12 kDa, which is far shorter than the heparin chains in native heparin proteoglycans. The macromolecular heparin molecule described in U.S. Pat. No. 5,529,986 is said to be especially useful for coating surfaces in extracorporeal circulation systems and its action is said to be based on binding to antithrombin III and enhancement of its activity, which is the main functional anticoagulant mechanism of all current heparin preparations.
Primarily, the standard heparin preparations are used for systemic treatment of thrombosis. As such they are most efficient in platelet-poor thrombi, such as venous thrombi, where coagulation activity prevails. The clinically used standard heparins, though effective in systemic treatment of thrombosis, by blocking the further growth of thrombosis, are not effective enough to prevent thrombotic complications, associated with either endogenous rupture of an atheromatous plaque or exogenous angioplasty or vascular or microvascular surgery.
Arterial interventions, such as angioplasty [PT(C)A=percutaneous transluminal (coronary) angioplasty] with or without stenting and vascular or microvascular surgery as well as (directional) arterectomy and peripheral thrombendarterectomy, represent a growing modality of treatment for cardiovascular diseases, which are the main cause of death. Accordingly, platelet-driven arterial thrombosis, which occurs in connection with endogenous vascular or microvascular injuries and/or exogenous interventions is a frequently encountered problem and in these situations the traditional systemic treatment of thrombosis is often of limited efficacy.
Current systemic antithrombotic treatment in connection with arterial interventions include the combination of an anticoagulant, such as unfractionated heparin (12 kDa) or low-molecular-weight heparins (7.5 kDa) with an antiplatelet drug, such as acetylsalicylic acid (cyclooxygenase inhibitor), or ticlopidine or better clopidogrel (ADP antagonist). The latest development is represented by potent platelet glycoprotein IIb/IIIa, von Willebrand factor and fibrinogen receptor antagonists, such as abciximab, tirofiban and velofibatide. The new combination treatments have succeeded in preventing 30-35% of acute thrombotic closures of the interventionally treated thrombus-prone vessels. The bleeding risk (major bleeding) requiring infusion of blood products is around 6-7%. So far abciximab has had the best efficacy, but since it is an antibody-based drug repetitive administration may cause antigenicity.
The systemic treatment with unfractionated heparin suffers from many unwanted effects, such as unpredictable bioavailability, short half-life, unspecific binding to proteins leading to compromised antithrombin III function and immunologic platelet effects with thrombocytopenia and thrombosis as well. These unwanted effects have been largely bypassed with the use of the low-molecular-weight, fractionated heparins, which however, have a limited capacity in inhibiting arterial thrombosis due to a limited control of fibrin-bound thrombin, and of platelet-bound factor Xa, and due to the neutralization of heparin-activity by platelet-secreted platelet factor 4. Thus, there is a great need for developing an effective and reliable prophylactic treatment of arterial thrombosis associated with vascular or microvascular injuries and interventions.
In their studies, the present inventors found that in contrast to the lower-molecular-weight heparins, native heparin proteoglycans (HEP-PG) obtainable from mammalian mast cells express potent antithrombotic properties, which are based on their capacity to inhibit platelet-collagen interactions. This unique property, namely blocking the platelet activation events subsequent to platelet adhesion to collagen, which is not present in the lower-molecular-weight, including unfractionated and fractionated commercial heparins, occurs simultaneously with a potential to enhance the function or activity of antithrombin III or heparin cofactor II.
In contrast to the traditional heparin mechanism, i.e. the antithrombin III enhancing action of heparins in the current clinical use as well as that of the macromolecular heparin construct described in U.S. Pat. No. 5,529,986, the efficacy of the heparin-like compounds of the present invention does not depend on the antithrombin III activity, but is based on a previously undescribed mechanism of strong inhibition of platelet activation triggered by platelet adhesion upon collagen. The exact mechanism is presently unknown, but is supposed to depend on disruption of the activation signal following platelet GP Ia/IIa binding to collagen and subsequent membrane phospholipid flip-flop and procoagulant activity, normally induced by collagen in platelets. Also, strong binding to von Willebrand factor (vWF) may be involved.
The present invention provides an alternative for antiplatelet treatment in form of local application, which can be combined with a systemic antiplatelet drug. This combination can be used in conjunction with angioplasty, vascular and microvascular surgery and endarterectomy to passivate the exposed subendothelial vascular and microvascular collagen for adhering platelets. During the initial studies, the desired effect achieved with the heparin proteoglycans (HEP-PG), was a significantly reduced local thrombus formation based on inhibition of platelet-to-platelet interaction, but preserved adhesion upon collagen. The collagen-induced platelet activation upon the adherent platelets was shown to be fully blocked in the presence of mast cell-derivable heparin proteoglycans (HEP-PG), multiple heparin glycosaminoglycan molecules, as such or connected to core molecules and lower-molecular-weight heparin or heparin-like glycosaminoglycans connected to spheroidal or globular core molecules to provide the spatially optimal macromolecular presentation or configuration which provides a sufficient coupling density of negatively charged glycosaminoglycans.
The above defined effect was obtainable both when the heparin glycosaminoglycan (HEP-GAG) having a molecular weight of 75±25 kDa and/or multiple carrier-(core molecule) coupled, spacer/linker-provided, unfractionated or fractionated heparin or heparin-like chains presented in the optimal spatial configuration and proteoglycan (HEP-PG) molecules comprising said HEP-GAG-molecules were in solution or immobilized on collagen-coated surfaces or administrated to vessel surfaces to be closed as anastomosis. Thrombin targeting with the current means to prevent thrombosis and to limit excessive wound repair, has still been associated with development of restenosis at the treated site. The advantage of the HEP-GAG- and HEP-PG-molecules of the present invention was preserved systemic platelet function which ensured normal hemostatic responses. The local inhibitory effect of mast cell-derived HEP-GAG- and HEP-PG-molecules on smooth muscle cell proliferation in vitro was also shown to be significantly better than that of lower-molecular-weight heparin species (Wang & Kovanen, Circulation Res, In Press).
Based on these preliminary findings the present inventors developed the heparin-like compounds of the present invention, as well as methods for their preparation and their use. The objectives of the invention are set forth below.
The first objective of the present invention was to provide soluble heparin-like compounds, mimicking the structure and properties of the mast cell-derived multiple heparin glycosaminoglycans (HEP-GAG) and/or heparin proteoglycans (HEP-PG), which HEP-GAG- and HEP-PG-molecules had been shown to be characterized by a hitherto unreported mechanism based on an almost complete inhibition of platelet-collagen interaction. Said mechanism is useful and convenient for screening and determining the properties of newly developed, synthetically, semisynthetically or biotechnologically modified heparin-like compounds as well as of locally applicable preparations useful for preventing thrombosis associated with vascular or microvascular injuries and interventions, such as angioplasty, stenting and vascular grafting.
Another objective of the invention was to provide pharmaceutically useful preparations, which comprise the heparin-like compounds of the present invention in combination with compatible adjuvants, carriers, etc.
A third objective of the invention was to provide means for and/or devices for administering the heparin-like compounds of the present invention by coating said means or devices.
The objective of the invention was also to provide methods for preparing said HEP-GAG- and HEP-PG-molecules from mast cells and to further modify the mast cells or the HEP-GAG- or HEP-PG-molecules by chemical and/or biotechnological methods to provide novel heparin-like compounds, with optimal spatial configurations and properties which are defined in the claims and which are similar to those of the native mast cell-derivable HEP-PG-molecules or the multiple HEP-GAG-molecules obtainable therefrom.
Still a further objective of the present invention was the use of the heparin-like compounds of the present invention as such or as ingredients for manufacturing preparations and devices useful for prophylactic treatment and prevention of severe vascular disorders including arterial thrombosis in connection with vascular and microvascular surgery or interventions, such as angioplasty, stenting and vascular grafting.